Effects of 4 testing arena sizes and 11 types of embryo media on sensorimotor behaviors in wild-type and mutant zebrafish larvae: Media and arena size impact zebrafish behavior.

The larval zebrafish is a highly versatile model across research disciplines, and the expanding use of behavioral analysis has contributed to many advances in neuro-psychiatric, developmental, and toxicological studies, often through large-scale chemical and genetic screens. In the absence of standardized approaches to larval zebrafish behavior analysis, however, it is critical to understand the impact on behavior of experimental variables such as the size of testing arenas and the choice of embryo medium. Using a custom-built, modular high-throughput testing system, we examined the effects of 4 testing arena sizes and 11 types of embryo media on conserved sensorimotor behaviors in zebrafish larvae. Our data show that testing arena size impacts acoustic startle sensitivity and kinematics as well as spontaneous locomotion and thigmotaxis, with fish tested in larger arenas displaying reduced startle sensitivity and increased locomotion. We also find that embryo media can dramatically affect startle sensitivity, kinematics, habituation, and pre-pulse inhibition, as well as spontaneous swimming, turning, and overall activity. Common media components such as methylene blue and high calcium concentration consistently reduced startle sensitivity and locomotion. To further address how the choice of embryo medium can impact phenotype expression in zebrafish models of disease, we reared mutant larvae, a model of CHARGE syndrome with previously characterized morphological and behavioral phenotypes, in 5 different types of media and observed impacts on all phenotypes. By defining the effects of these key extrinsic factors on larval zebrafish behavior, these data can help researchers select the most appropriate conditions for their specific research questions, particularly for genetic and chemical screens.